Project 1: Gene Therapy for Adenosine Deaminase-deficient Severe Combined Immunodeficiency (ADA-SCID). Traditional forms of treatment for ADA-SCID (HSCT and enzyme replacement therapy, ERT) are either not available to all patients or have unsatisfactory results. Corrective gene transfer has therefore been developed to provide an alternative form of treatment for this disease. In 2000, we begun a gene therapy trial for ADA deficient patients on ERT using two new retroviral vectors that express ADA under the control of the myeloproliferative sarcoma virus (MPSV) LTR in wild type configuration or containing the MND modifications of the negative control region (ncr) and primer binding site (pbs) that have been demonstrated to confer higher resistance to methylation-mediated gene silencing in murine models. The results from four patients enrolled in this trial in collaboration with Dr. Donald B. Kohn at Children's Hospital Los Angeles and University of California Los Angeles, showed long-term, but low-level of gene-corrected cells in only two patients with no evident immunological/metabolic improvement. We amended the trial to test the hypothesis that using preparative chemotherapy in the absence of ERT would afford a better outcome. From November 2005 to March 2009, we enrolled six patients who underwent withdrawal of ERT and administration of low-dose busulfan (75 mg/m2) before reinfusion of gene-corrected cells. The results from this trial reveal clear evidence of immune reconstitution three out of six patients and increased marking by the MND vector (Candotti F., Shaw K.L., Muul L., Carbonaro D., Sokolic R., Choi C., Schurman S.H., Garabedian E., KesserwanC., Jagadeesh G.J., Fu P-Y., Gschweng E., Cooper A., Tisdale J.F., Weinberg K.I., Crooks G.M., Kapoor N., Shah A., Abdel-Azim H., Yu X-J., Smogorzewska M., Wayne A.S., Rosenblatt H.M., Davis C.M., Hanson C., Rishi R.G., Wang X., Gjertson D., Yang O.O., Balamurugan A., Bauer G., Ireland J.A., Engel B.C., Podsakoff G.M., Hershfield M.S., Blaese R.M., Parkman R., Kohn D.B: Gene Therapy for Adenosine Deaminase-Deficient Severe Combined Immune Deficiency: Clinical Comparison of Retroviral Vectors and Treatment Plans. Blood, 2012;120:3635). These results, however, differ from the Milan experience where a faster immune reconstitution was observed in eight out of ten treated patients. We postulate that the overall higher dose of busulfan used in the Italian trial and the larger number of infused cells may explain the difference. To test this hypothesis, we have increased the busulfan to 90 mg/m2 in the two most recent patients and have used dosage for the next series of 10 patients to evaluate if a prompter engraftment of gene-corrected cells can be achieved with these conditions. In addition, because of the preliminary evidence of the superior marking afforded by the MND vector, the next phase of the trial will only administer cells transduced with such vector. Increasing the bone marrow collection volume would likely result in higher numbers of reinfusable cells, however, it would also increase morbidity for our patients. As an alternative option to achieve higher numbers of reinfused gene-corrected cells, we will test the use of lentiviral vectors that can be hypothesized to translate in higher transduction efficiency of HSCs. In collaboration with Drs. Donald Kohn and Bobby Gaspar (UCL, London), we are testing the efficacy of an ADA-expressing lentiviral vector (LV) in a 3-site international clinical trial that opened in the USA in the summer of 2013 and enrolled 6 patients to date. Project 2: Gene Therapy for Wiskott-Aldrich Syndrome (WAS). HSCT from HLA-identical donors can cure all aspects of WAS, but it is only available to 15% of the patients. We and others have therefore proposed gene therapy as a potential useful alternative treatment and demonstrated that WAS gene transfer can correct the biological defects observed in patients lymphocytes and Was KO animals.These extensive pre-clinical results paved the way to the first clinical trial in Germany that used a retroviral vector. Unfortunately, insertional oncogenesis events occurred in this trial that resemble those occurred the French and UK gene therapy trials for X-SCID. To increased safety of gene transfer protocols for WAS, several groups have developed LVs for gene therapy of WAS. We have tested Foamy virus-based WASp-expressing vectors and demonstrated that they restore lymphocyte and platelet function in a mouse model of WAS (Uchiyama T., Adriani M., Jagadeesh G.J., Paine A., Candotti F.: Foamy virus vector-mediated gene correction of a mouse model of Wiskott-Aldrich syndrome. Mol Ther 2012; 20:1270-9). Any gene transfer system based on integrating viral vectors has the general limitations of gene addition methods, such as eviction from physiological control of gene expression and potential detrimental effects from the persistence of the mutated protein expression. The possibility of targeted repair of the disease-causing mutation remains therefore the ultimate goal of gene therapy. Increasingly applied strategies for gene editing and repair are based on the use of Zinc Finger Nucleases (ZFNs) and the CRISPR/Cas-9 systems that can be engineered to virtually recognize any desired sequence and exploited to induce gene repair, gene targeting and gene deletion. We have begun testing the hypothesis that the precise insertion of the cDNA sequences corresponding the WAS exons 2-12 within the WAS intron 1 genomic sequence will provide a general gene-repair strategy applicable to all patient mutations occurring within those exons. Mouse ES cells containing a Was gene-disrupting insertion in exon 7 (Was KO) have been transfected with a ZFN or CRISPR/Cas-9 molecules specific for intron 1 sequences together with the exon 2-12 donor cDNA fragment flanked by intron 1 and 2 homology segments. Cells in which successful recombination occurred were selected and injected into fertilized blastocysts for production of gene-repaired animals and generation of proof-of-principle data that will support the application of gene editing strategies in humans.